The present invention concerns a new method for in vivo labeling of biopolymers, like proteins, nucleic acids, lipids, carbohydrates, and biodegradable plastic, with isotopes, especially stable isotopes and the use of chemolithotrophic bacteria for in vivo isotope labeling of biopolymers. The invention concerns in particular the use of CO2-fixing bacteria, like Ralstonia eutropha and similar methanogenic bacteria for in vivo isotope labeling, especially for labeling with the stable isotope 3C and the use of isotope-labeled biomolecules in therapeutic and diagnostic applications, especially in spectroscopy methods and generally as tracer compounds.
The deliberate use of biomolecules in diagnosis and therapy requires knowledge of the structure and dynamics of these molecules. This information can be obtained, for example, by using nuclear magnetic resonance (NMR). A prerequisite for efficient use of this method, however, is labeling of the biomolecules of interest with stable isotopes, so-called S isotopes, like 2H, 13C and 15N.
Since S isotopes are not radioactive and, except for deuterium, are not toxic either, they have considerable diagnostic potential. S isotopes have already been successfully used in metabolic diagnosis. In addition to 2H, 13C and 15N, the nuclei 1H, 7Li, 11B, 14N, 17O, 18O, 19F, 23Na, 29Si, 31P, 33S and 77Se have been used above all for NMR spectroscopy of organic compounds (cf., Vogel, H. J. (1989), Methods in Enzymology, Vol. 177, 263).
Use of S isotope-labeled substances, especially labeled amino acids, is discussed in imaging NMR spectroscopy, especially NMR microscopy. With further development of imaging NMR methods, the area of application of S isotope-labeled substances will expand and will not be restricted merely to the aforementioned S isotopes 2H, 1C and 15N.
Thus far, complete S isotope-labeling of proteins and other biopolymers has generally occurred by in vivo labeling, i.e., organisms are cultured on isotope-labeled media and the desired protein or another component is then isolated from the labeled organisms.
The medium necessary for culturing, which generally consists of a carbon source, nitrogen source and salts, is more demanding and more cost-intensive, the more complex it is. This is particularly true of the carbon source. Since nutrient media for higher, i.e., eukaryotic, organisms are particularly complex, expression of S isotope-labeled eukaryotic proteins mostly occurs in recombinant fashion in bacteria, in most cases in Escherichia coli (see, for example, Donne, D. G. et al. (1997), Proc. Natl. Acad. Sci. USA 94, 13452-13457). These bacteria use glucose as inexpensive 13C-labeled carbon source. 13C-labeled methanol, which is relatively cheap, has been used as an alternative to culture Methylophilus methylotrophus (Batey, R. et al. (1995), Methods in Enzymology 261, 300-322). The most cost-effective carbon source is 13C-labeled CO2. Green algae (for example, Chlorella vulgaris, Chlorella pyrenoidosa, Chlorella fusca or Scenedesmus obliquus) can fix carbon dioxide by photosynthetic reduction and therefore be completely labeled by feeding of 13C-labeled CO2. The labeled algal hydrolyzate is then reused as carbon source to culture bacteria, especially E. coli. 
The use of algal hydrolyzate instead of glucose is not only more economical, but in many cases also necessary for biological reasons. If foreign proteins are expressed in E. coli, the attainable cell density often drops, especially if minimal medium with glucose is used as carbon source. In many cases, the expression of a heterologous protein in E. coli is only possible by using algal hydrolyzate as C source; often at least the yield of heterologously expressed protein and thus the efficiency of the labeling method can be increased by supplying algal hydrolyzate.
Plasmid DNA from E. coli is manipulated for production of S isotope-labeled nucleic acids, especially DNA so that the desired nucleic acid sequence is displayed on the plasmid. An alternative method, which is obligatory for RNA and optional for DNA, is isolation of the entire RNA and DNA from labeled cells (E. coli or algae). After hydrolysis of DNA or RNA, isolation of nucleotides and phosphorylation to ribonucleoside triphosphates or deoxyribonucleoside triphosphates, new nucleic acids are synthesized in vitro with appropriate polymerases (Mer, G. and Chazin, W. J. (1998), J. Am. Chem. Soc. 120, 607-608).
Labeling with 13C occurs in the prior art by means of complex and therefore costly carbon sources like 13C-glucose in the case of in vivo labeling of bacteria, like E. coli, or alternatively, using 13C-labeled carbon dioxide in the case of in vivo labeling of green algae and then supplying the 13C labeled algal hydrolyzate as C source.
Labeling with 15N generally occurs by using correspondingly labeled salts, labeling with 2H by culturing in D2O. Labeling with other isotopes occurs in similar fashion by metabolism of appropriately labeled substances.
In many cases labeling with several S isotopes is necessary. Double or multiple labeling can be achieved by a combination of different methods.
Radioactively-labeled biopolymers also find numerous applications in therapy and diagnosis. Thus, biomolecules labeled with radioactive isotopes are used, for example, as tracers or markers in testing metabolic and circulatory functions, in following and visually examining enrichment processes in tissues and organs, in radioimmunological and related in vitro assay methods as radiopharmaceuticals in nuclear medical in vivo diagnosis (for example, scintigraphy) and in autoradiography.
One task of the invention is to provide a new method for in vivo labeling of molecules a with isotopes.
A special task is to provide a process for in vivo labeling of biopolymers with stable isotopes, especially 13C, alone and in combination with other isotopes.
Another task consists of providing an in vivo labeling method that overcomes the drawbacks of the prior art methods and can be conducted more efficiently and more cost effectively in comparison with known methods.
These and other tasks are solved by the method according to the invention, which is based on the use of chemolithotrophic bacteria and is defined in the independent claims. Preferred variants of the invention are apparent from the subclaims.
In a particular variant of the method, in vivo labeling of biopolymers with isotopes, preferably stable isotopes, occurs by means of CO2-fixing bacteria, labeling occurring in a preferred variant with stable and/or unstable carbon isotopes in methanogenic bacteria. Examples of methanogenic bacteria that are suitable for the in vivo labeling method according to the invention include Acidovorax facilis, Alcaligenes ruhlandii, Alcaligenes latus, Alcaligenes sp. 2625, Ancylobacter aquaticus, Ancylobacter sp. 1106-1108, 2456, 2457, 2666-2669, Aquifex pyrophilus, Aquaspirillum autotrophicum, Azospirillum amazonense, Azospirrillum sp. 1726, 1727, Azospirilum lipoferum, Azotobacter sp. 1721-1723, Bacillus schlegelii, Bradyrhizobium japononicum, Bacillus tusciae, Calderobacterium hydrogenophilum, Campylobacter sp. 806, Derxia gummosa, Hydrogenophaga flava, Hydrogenobacter thermophilus, Hydrogenophaga palleronii, Hydrophaga pseudoflava, Hydrogenophaga taeniospiralis, Mycobacterium gordonae, Oligotropha carboxidovorans, Paracoccus denitrificans, Pseudomonas saccharophila, Pseudocardia autotrophica, Pseudocardia petroleophila, Pseudocardia saturnea, Ralstonia eutropha, Variovorax paradoxus, Xanthobacter agilis, Xanthobacter autotrophicus and Xanthobacter flavus. Labeling occurs with particular preference in Ralstonia eutropha (previously also named Alcaligines eutropha).